LOCUS       sequence-296870-        8500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bioluminescent reporter consisting of a membrane-bound SEP and 
            Nanoluc fusion; for monitoring extracellular tumor acidosis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8500)
  AUTHORS   Ong TT, Ang Z, Verma R, Koean R, Tam JKC, Ding JL
  TITLE     pHLuc, a Ratiometric Luminescent Reporter for in vivo Monitoring of 
            Tumor Acidosis.
  JOURNAL   Front Bioeng Biotechnol. 2020 May 8;8:412. doi: 
            10.3389/fbioe.2020.00412. eCollection 2020.
  PUBMED    32457886
REFERENCE   2  (bases 1 to 8500)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Front 
            Bioeng Biotechnol."; date: "2020-05-8"; volume: "8:412. doi"; pages:
            " 10.3389/fbioe.2020.00412. eCollection 2020"
COMMENT     Sequence Label: SEPLuc
FEATURES             Location/Qualifiers
     source          1..8500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             10..63
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             70..669
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(70..89)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     566..586
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    682..1270
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(735..755)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1153..1164)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1344..1577
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1649..1770
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1686..1705)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1740..1759
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1789..1924
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1957..1973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1957..1973)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1970..1992)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1981..1997
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2005..2011,2012..2029,2030..2035))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2050..2071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2188..2205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2359..2947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(3118..3909,3910..3978))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3741..3760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3979..4083)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4151..4169
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4207..4229)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4329..4348
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4542..4564
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4556..4573
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4557..4573
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4588..4814
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             4815..4995
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5042..5167
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5660..5893
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6078..6122
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    6389..6505
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6548..6759
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             6772..7040
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
ORIGIN
        1 gcggccgctg agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc
       61 ccttccggga tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
      121 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
      181 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
      241 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
      301 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
      361 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
      421 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
      481 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
      541 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
      601 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
      661 ggtgcctgaa tctaggtcga caatcaacct ctggattaca aaatttgtga aagattgact
      721 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
      781 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
      841 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
      901 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
      961 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     1021 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     1081 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     1141 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     1201 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     1261 gcctccccgc ctggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     1321 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gataagatct
     1381 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     1441 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     1501 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     1561 gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc
     1621 aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa
     1681 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     1741 tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta
     1801 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     1861 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     1921 ggcctagact tttgcagaga ccaaattcgt aatcatgtca tagctgtttc ctgtgtgaaa
     1981 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     2041 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     2101 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     2161 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     2221 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     2281 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2341 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2401 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2461 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2521 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     2581 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2641 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2701 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2761 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     2821 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     2881 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     2941 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3001 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3061 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3121 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3181 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3241 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     3301 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3361 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3421 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3481 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3541 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3601 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3661 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3721 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     3781 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3841 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3901 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3961 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4021 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     4081 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     4141 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     4201 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     4261 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     4321 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     4381 gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa
     4441 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     4501 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     4561 aacgacggcc agtgccaagc tgacgcgtgt agtcttatgc aatactcttg tagtcttgca
     4621 acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc
     4681 gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga
     4741 catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag
     4801 ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct
     4861 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     4921 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     4981 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
     5041 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
     5101 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
     5161 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
     5221 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
     5281 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
     5341 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
     5401 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
     5461 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggcca ctgatcttca
     5521 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     5581 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     5641 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     5701 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     5761 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     5821 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     5881 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     5941 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     6001 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     6061 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     6121 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     6181 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     6241 ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca
     6301 ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag
     6361 tgaacggatc tcgacggtat cggttaactt ttaaaagaaa aggggggatt ggggggtaca
     6421 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     6481 aacaaattac aaaattcaaa attttatcga tactagtgga tctgcgatcg ctccggtgcc
     6541 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     6601 aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     6661 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     6721 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg aggggctcgc
     6781 atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg gttgagtcgc
     6841 gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta ggtaagttta
     6901 aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc tagactcagc
     6961 cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt ttcgttttct
     7021 gttctgcgcc gttacagatc caagctgtga ccggcgccta ctctagagca tggttgagat
     7081 gttgccaacc gtagctgtgc tcgttttggc tgtcagcgta gtcgctaaag ataatacaac
     7141 acttcaggag ttcgcaacat ctaagggaga agagttgttt actggcgtag tacccattct
     7201 tgtggaactt gacggcgatg ttaatggtca taagttctct gtgtctggtg agggcgaggg
     7261 ggatgctaca tatggtaaat tgaccctcaa atttatttgt acaaccggga agctccctgt
     7321 accctggcct actctcgtga ccaccctcac ctacggggta caatgttttt ccagataccc
     7381 cgatcacatg aagcgccacg atttcttcaa aagtgccatg cccgagggtt acgtccagga
     7441 acgcacaata tttttcaagg atgatggaaa ttacaaaacc cgcgccgagg ttaagttcga
     7501 gggggatacc ctcgttaacc ggattgaact gaagggtatc gactttaagg aggacggtaa
     7561 tatattgggg cataaattgg aatacaacta taacgaccac caggtgtata tcatggccga
     7621 taaacagaag aacggcatca aagcaaactt taaaattagg cataacatag aggatggtgg
     7681 cgttcagctt gctgatcact atcaacagaa tacccctata ggtgatggac ccgtgctcct
     7741 cccagataac cattacttgt ttacaacttc tacactctct aaggacccca acgaaaagag
     7801 agaccacatg gtgctcttgg agttcgtaac tgccgccgga attacacatg gtatggacga
     7861 gctctacaag gatatttccg ggggggtctt tacacttgaa gatttcgtcg gggattggag
     7921 acaaactgcc ggttataatc ttgatcaggt gcttgagcaa gggggggtga gctccctttt
     7981 tcaaaacctg ggtgtatctg tcacccctat ccaaaggatt gtgcttagcg gtgaaaatgg
     8041 tctcaaaatc gacatccacg tgatcatccc atacgagggt cttagtggag atcagatggg
     8101 gcagatagag aagattttca aagttgttta ccctgtcgac gaccaccatt tcaaagtaat
     8161 tctgcactac ggaaccctcg tcatagacgg ggtcacaccc aacatgatag attactttgg
     8221 gcgaccctat gaagggatag cagtattcga cggcaaaaag ataaccgtga caggcaccct
     8281 gtggaatgga aacaaaatta tagatgagcg cctgatcaac cctgacggtt ctttgttgtt
     8341 cagggttaca atcaatggtg tcacaggctg gaggctctgt gagcgcatcc tggctcagtt
     8401 ggagaatggg gggatctccc tcttggtgca aaatacatca tggatgttgc ttcttcttct
     8461 tagcttgagc ctcttgcagg cattggattt catcagtctc
//
